Traditional therapeutic molecules circulate freely throughout the body of patients until they are removed from circulation by the liver or another mechanism of clearance. Such non-targeted molecules can exert pharmacological effects indiscriminately on a wide range of cells and tissues. Indiscriminate targeting can cause serious side effects in a patient. The problem may be particularly acute when the molecule is highly toxic (e.g., in the case of a chemotherapeutic agent where the therapeutic window, the difference between an efficacious and injurious or even lethal dose, can be small).
In recent years, researchers have attempted to develop compounds that specifically affect particular groups of cells, tissues or organs. Most of the compounds target a particular tissue by preferentially binding a particular target molecule displayed by the tissue. By preferentially affecting targeted cells, tissues or organs, the therapeutic window can be increased, which in turn increases the opportunity for a successful treatment regimen and/or reduces the occurrence of side effects.
Preferential binding is employed in antibody-directed enzyme prodrug therapy (ADEPT). See, e.g., Xu et al., 2001, Clin Cancer Res. 7:3314-24.; Denny, 2001, Eur J Med Chem. 36:577-95. In ADEPT, an antibody or antibody fragment is linked to an enzyme capable of converting an inactive pro-drug into an active cytotoxic agent. An ADEPT conjugate is administered to a patient, and the conjugate is localized to a target tissue via antibody/antigen binding. The prodrug is subsequently administered, and the prodrug circulates throughout the patient's body but causes few or no side effects because the prodrug is in the inactive form and is activated by the ADEPT antibody-enzyme conjugate only in the vicinity of the target tissue. Thus, a relatively low concentration of active drug is present throughout the body, but a relatively high concentration of active drug is produced in the vicinity of the target tissue, increasing the therapeutic window of the toxin at the desired site.
In ADEPT, the antibody or antibody portion of a construct binds to an antigen to achieve localization, so selecting the proper antigen is important (e.g., an antigen that has a high tumor/normal expression profile). An antigen of particular interest frequently found on the cell surface in cancer tissues is carcinoembryonic antigen (CEA). CEA was first described by Gold and Freedman, J. Exp. Med., 121, 439-462, (1965). CEA is highly expressed in tumor tissue and also found at a lower concentration in some normal organs, particularly in the digestive tract.
Many antibodies to tumor antigens cross-react with related antigens. Systemic application of a MAb that is cross-reactive with a related antigen must be avoided to preclude risk of potentially severe side effects. Accordingly, the development of antigen-specific monoclonal antibodies for in vitro and in vivo diagnosis and therapy requires a good knowledge of the number, quality and biodistribution of related cross-reactive antigens. Careful immunochemical characterization of the MAb to be used is required with respect to its specificity and affinity for the target antigen and for related antigens.
Murine MAb T84.66 (ATCC Accession No. BH 8747) IgG1, k shows a high affinity constant to CEA and no cross reactivity to other members of the CEA gene family. A significant potential side effect of ADEPT therapy is the development of antibodies against the targeted enzyme during therapy. The production of human anti-mouse antibodies (HAMA) leads to reduced efficiency of the MAb and to potentially serious manifestations of acute and chronic allergic complications for the patient. See Levy, et al. Ann. Rev. Med. 34:107-116 (1983); Houghton, et al. Proc. Natl. Acad. Sci. USA, 82:1242-1246 (1985) and Sears, et al. J. Biol. Resp. Modifiers 3:138-150 (1984). Antibody formation has been observed during a clinical trial of a CEA-directed antibody-enzyme conjugate two weeks after treatment, which prevented subsequent rounds of treatment [Napier, M. P., S. K. Sharma, C. J. Springer, K. D. Bagshawe, A. J. Green, J. Martin, S. M. Stribbling, N. Cushen, D. O'Malley and R. H. Begent (2000) Clin Cancer Res 6, 765-72, Antibody-directed enzyme prodrug therapy: efficacy and mechanism of action in colorectal carcinoma].
The risk of eliciting an immune reaction is high for microbial proteins. The use of human enzymes for ADEPT has been investigated in pre-clinical studies [Smith, G. K., S. Banks, T. A. Blumenkopf, M. Cory, J. Humphreys, R. M. Laethem, J. Miller, C. P. Moxham, R. Mullin, P. H. Ray, L. M. Walton and L. A. Wolfe, 3rd (1997) J Biol Chem 272, 15804-16, Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate]. Although the risk of antibody formation can be reduced for human protein as compared to microbial protein, human proteins can also elicit immune reactions when administered to people. The consequences of eliciting an immune reaction against a human protein can be very significant, as such a treatment could trigger an auto-immune disease.
The risk of eciciting an immune reaction may be great for an ADEPT construct that contains at least two potential risks: the antibody portion and the enzyme portion.